EP0734731A2 - Vaccins composés de constituants multiples contenant des organismes clostridium et non-clostridium à faible dose - Google Patents

Vaccins composés de constituants multiples contenant des organismes clostridium et non-clostridium à faible dose Download PDF

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Publication number
EP0734731A2
EP0734731A2 EP96104209A EP96104209A EP0734731A2 EP 0734731 A2 EP0734731 A2 EP 0734731A2 EP 96104209 A EP96104209 A EP 96104209A EP 96104209 A EP96104209 A EP 96104209A EP 0734731 A2 EP0734731 A2 EP 0734731A2
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Prior art keywords
clostridial
vaccine
protective antigen
virus
adjuvant
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English (en)
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EP0734731A3 (fr
EP0734731B1 (fr
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Richard E. Parizek
Lonny E. Vlieger
Sharon A. Bryant
Stuart K. Nibbelink
Michael J. Mcginley
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Bayer Corp
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Bayer AG
Bayer Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to low dose multicomponent vaccines. More specifically, the invention relates to low dose multicomponent vaccines comprising a safe and immunogenically effective combination of: at least one protective antigen component from clostridial organisms, at least one protective antigen component from a non-clostridial organism and an adjuvant.
  • Multicomponent vaccines of interest are those vaccines that contain as essential antigen components: one or more protective antigens from one or more organisms and an adjuvant.
  • the protective antigen component can be in the form of a whole bacterial culture, a whole virus culture, a cell-free toxoid, a purified toxoid, or a subunit.
  • the formulation When one combines whole cultures of organisms (viruses or bacteria) in a formulation of multicomponent vaccines, the formulation would contain numerous antigens (hundreds to thousands). Some of these are protective antigens as mentioned above. Some of these antigens are detrimental to protection of the animals or cause reaction in the animals ("detrimental antigens"). The detrimental antigens can interfere with the protective antigens by either physically or chemically blocking the active sites of the protective antigens. The interference prevents the protective antigens from protecting animals. Also, the detrimental antigens can produce negative responses such as local reactions, systemic reactions, anaphylaxis and/or immunosuppression in the animals. Therefore, the use of combinations of whole culture organisms can cause problems with efficacy or with animal reactivity. Animal reactivity produces localized reactions resulting in swellings or abscesses at the injection sites or a systemic response such as anaphylaxis that can result in death of the animal.
  • Aggravating the animal reactivity is the administration of multicomponent vaccines to large animals (e.g., cattle) in high doses.
  • the dose range has historically been from about 5 mL to 10 mL to allow incorporation of all of the protective antigens into one formulation.
  • up to seven clostridial whole cultures or toxoids can be combined into a 5.0 mL dose of vaccine for administration to cattle. See, for instance, pages 319, 320, 321, 322, and 432 of the Compendium of Veterinary Products, Third Edition, 1995-1996).
  • 6 Clostridial whole cultures or toxoids have been combined with Hemophilus somnus in a 5.0 mL dose vaccines. See pages 191, 192, 319, 433, 490, and 1013 of the Compendium of Veterinary Products, Third Edition, 1995-1996). Reportedly, such vaccines demonstrate significant animal reactivity.
  • the injection site lesions must be cut out of the meat and discarded. This causes significant monetary loses to retailers, beef packers and feedlots. It has been estimated that 12-15% of prime beef cuts have some type of injection site lesion that must be trimmed away (Effertz, Beef Today, March 1991). This article attributes the main cause of the injection site lesions to 7-way clostridial vaccines. Additionally, there have been reports that up to 90% of cattle have injection site lesions in their carcass.
  • Injection site lesions have been associated with: (1) the presence of many detrimental antigens or contaminants which are present in whole culture vaccines, (2) the adjuvants incorporated into such vaccines, (3) the method of administration of such vaccines (4) the large dose size of some of the multicomponent vaccines (5.0 - 10.0 mL), and (5) animal the reactivity of the protective antigen components of the vaccines.
  • clostridial vaccines are not highly purified because purification can be cost prohibitive.
  • animal vaccine production must be necessarily economically effective if the vaccines are to enjoy widespread use. Therefore, highly purified animal vaccines are virtually cost prohibitive.
  • Antigenic components of clostridial vaccines were typically obtained by concentrating whole cultures of the bacteria. Concentration was accomplished by precipitating whole cultures with ammonium salts such as ammonium sulfate or concentrating such whole cultures via ultrafiltration. Both procedures are costly. Additionally, these procedures produce massive amounts of cells resulting in a high antigen mass that remains as an antigenic mass of solids in the product. Such a high antigenic mass would induce animal reactivity, particularly injection site lesions.
  • the vaccines must contain protective antigens described herein. While one could administer each of the protective antigens in a monovalent vaccine, this mode of administration would require several vaccinations for each animal. This is impractical in a because: 1) handling animals for repeated vaccinations can result in undue stress and consequential diseases; 2) labor for performing such vaccinations is expensive compared to the profit obtained from each animal; 3) the more injection sites on an animal, the more potential for injection site reactions.
  • low dose multicomponent vaccines containing: protective antigen components of a clostridial organism(s) and at least one non-clostridial protective antigen component and an adjuvant, and the processes for making and using the vaccines.
  • This invention relates to a multicomponent vaccine comprising: a safe and immunogenically effective combination of protective antigen components from at least one clostridial organism, a protective antigen component from a non-clostridial organism and an adjuvant, wherein the vaccine is in a low dose volume.
  • low dose is meant dose volumes, including the adjuvant which are less than 5.0 mL and which do not adversely affect the protective antigen components or the animal post vaccination.
  • an antigen is that which produces an antibody response against the antigen, which response is not necessarily protective.
  • protecting antigen is meant an antigen that produces an immune response and imparts protection to the animal.
  • a vaccine containing such a protective antigen is characterized as "immunogenically effective.”
  • a multicomponent vaccine for ruminants comprising: a safe and immunogenically effective combination of a protective antigen component from at least two and preferably six to seven clostridial organisms; a protective antigen component from a non-clostridial organism and an adjuvant, wherein the vaccine is in a low dose volume.
  • the multicomponent vaccine comprises a safe and immunogenically effective combination of an antigen component from one or more clostridial organisms; an antigen component from an organism selected from the group consisting of a Gram negative organism, a Gram positive organism, a virus, a parasite and a rickettsia and an adjuvant wherein the vaccine is in a dose size of 3.0 mL or less.
  • the multicomponent vaccine for ruminants comprises a safe and immunogenically effective combination of an antigenic component from six clostridial organisms, which are Clostridium chauvoei , Clostridium septicum , Clostridium novyi , Clostridium perfringens type C, Clostridium perfringens type D and Clostridium sordellii , an antigen component from H. somnus or M. bovis and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
  • six clostridial organisms which are Clostridium chauvoei , Clostridium septicum , Clostridium novyi , Clostridium perfringens type C, Clostridium perfringens type D and Clostridium sordellii , an antigen component from H. somnus or M. bovis and an adjuvant, wherein the vaccine is in a dose size of 3.
  • the multicomponent vaccine for ruminants comprises: a safe and immunogenically effective combination of a protective antigen component from seven clostridial organisms which are Cl. chauvoei , Cl. septicum , Cl. novyi , Cl. perfringens type C, Cl. perfringens , type D, Cl sordellii , and Cl. haemolyticum ; an antigen component from Haemophilus somnus or Moraxella bovis and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
  • a protective antigen component from seven clostridial organisms which are Cl. chauvoei , Cl. septicum , Cl. novyi , Cl. perfringens type C, Cl. perfringens , type D, Cl sordellii , and Cl. haemolyticum ; an antigen component from Haemophilus somnus or Moraxella bovis and
  • the multicomponent vaccine for ruminants comprises: a safe and immunogenically effective combination of an antigen component from at least two clostridial organisms such as Cl. perfringens type C and Cl. perfringens type D; an antigen component from a virus such as an infectious bovine rhinotracheitis virus (IBRV) and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
  • an antigen component from at least two clostridial organisms such as Cl. perfringens type C and Cl. perfringens type D
  • an antigen component from a virus such as an infectious bovine rhinotracheitis virus (IBRV) and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
  • IBRV infectious bovine rhinotracheitis virus
  • a particularly preferred embodiment of this invention includes a multicomponent vaccine for ruminants comprising: a safe and immunogenically effective combination of a protective antigen component from more than two clostridial organisms selected from the group consisting of Cl. chauvoei , Cl. septicum , Cl. novyi , Cl. perfringens type C, Cl. perfringens type D, Cl sordellii , and Cl.
  • haemolyticum protective antigen components from viruses which are selected from the group consisting of an infectious bovine rhinotracheitis virus (IBRV), a parainfluenza type 3 virus (PI 3 V), a bovine virus diarrhea virus (BVDV) and a bovine respiratory syncytial virus (BRSV) and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
  • IBRV infectious bovine rhinotracheitis virus
  • PI 3 V parainfluenza type 3 virus
  • BVDV bovine virus diarrhea virus
  • BRSV bovine respiratory syncytial virus
  • the multicomponent vaccine comprises: a safe and immunogenically effective combination of a protective antigen component from at least six clostridial organisms; a protective antigen component from a plurality of viruses and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
  • the most preferred embodiment of the invention is a multicomponent vaccine comprising: a safe and immunogenically effective combination of a protective antigen component from at least seven clostridial organisms; protective antigen components from at least four viruses and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
  • a method for producing a multicomponent vaccine comprising a safe and immunogenically effective combination of protective antigen components from clostridial organisms and a protective antigen component from a non-clostridial organism and an adjuvant wherein the vaccine is in a dose size of 3.0 mL or less, said method comprising: 1) identifying the protective antigen component of each organism by in vivo or in vitro methods; 2) quantitating the protective antigen components using antigen quantitation assays to provide the protective antigen component in an amount sufficient to produce a protective vaccine with the least antigenic mass; 3) identifying components of the organisms containing detrimental antigens by using the antigen quantitation assays and animal reactivity testing; 4) purifying the protective antigen components which contain detrimental antigens to remove the detrimental antigens; 5) selecting for each organism requiring inactivation, an effective inactivating agent which kills the organism without denaturing the protective antigen component; 6) selecting an effective adjuvant which produces enhancement of immune
  • encompassed by the invention is a process for administering the vaccines of the invention to ruminants.
  • the present invention it has been demonstrated that there is a significant difference in the size of injection site lesions in cattle vaccinated with: (1) a conventional 5.0 mL dose multicomponent clostridial product and (2) the low dose (2.0 mL) multicomponent vaccine of this invention.
  • the area of the injection site lesion produced by the low dose vaccine is significantly smaller, post injection than the lesion produced by the conventional 5.0 mL dose vaccine.
  • the low dose multicomponent vaccine produced injection site lesions in an insignificant number of cattle as compared with the conventional vaccine.
  • the invention relates to a multicomponent vaccine comprising a safe and immunogenically effective combination of: an antigen component from one or more clostridial organisms; an antigen component from a non-clostridial organism selected from the group consisting of a Gram negative organism, a Gram positive organism, a virus, a parasite and a rickettsia and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
  • Non-limiting examples of the clostridial organisms and diseases in ruminants are as follows:
  • Clostridium chauvoei causes the disease blackleg.
  • This organism like all Clostridial organisms, produces spores that can survive in soil for years and, during this time, can infect susceptible animals (cattle and sheep) which ingest them.
  • Blackleg is an acute, infectious but noncontagious, disease of cattle and sheep characterized by gaseous tissue swelling, usually in the heavy muscles. The organism enters cattle or sheep via feed or cuts or by shearing, docking, or castration. The onset of the disease is quite sudden. Body temperature rises rapidly and muscular stiffness, depression and reluctance to move are prominent. When infection is extensive, death often occurs within 16-72 hours. Treatment of sick animals is futile since there is often permanent damage done to their meat.
  • the non-clostridial organism can be selected from the group consisting of: a Gram negative organism, a Gram positive organism, a virus, a parasite and a rickettsia.
  • a Gram negative organism a Gram positive organism
  • a virus a virus
  • a parasite a parasite
  • a rickettsia a non-limiting illustration of the Gram negative organisms.
  • clostridial organisms can be selected from the group consisting of: Cl. chauvoei , Cl. septicum , Cl. novyi , Cl. perfringens type C, Cl. perfringens type D, Cl sordellii , and Cl. haemolyticum .
  • the protective antigen of the clostridial component is derived from six to seven clostridial organisms.
  • the non-clostridial protective antigen component can be selected from the group consisting of Gram negative bacteria, Gram positive bacteria, viruses, parasites, rickettsia and a combination thereof.
  • Gram negative organisms can be selected from the group consisting of: H. somnus , M. bovis , E. coli , Salmonella typhimurium , Pasteurella hemolytica , Pasteurella multocida , Campylobacter fetus , Leptospira spp and a combination thereof.
  • Preferred herein are H. somnus and M. bovis .
  • Non-limiting examples of the Gram positive organisms are Clostridium tetani , Bacillus anthracis , Listeria monocytogenes , Actinomyces pyogenes and a combination thereof.
  • Non-limiting examples of the virus can be selected from the group consisting of: infectious bovine rhinotracheitis (IBRV), parainfluenza virus type 3 (PI 3 V), bovine virus diarrhea virus (BVDV) bovine respiratory syncytial virus (BRSV) and a combination thereof.
  • IBRV infectious bovine rhinotracheitis
  • PI 3 V parainfluenza virus type 3
  • BVDV bovine virus diarrhea virus
  • BRSV bovine respiratory syncytial virus
  • Non-limiting examples of the parasites are Neospora spp. , Tritrichimonas foetus , Cryptosporidia spp. and a combination thereof.
  • a non-limiting example of the rickettsia is Ehrlichia bovis .
  • the clostridial and non-clostridial protective antigen components can be in the form of: inactivated or modified live whole cultures, toxoids, cell-free toxoids, purified toxoids, subunits or combinations thereof.
  • Adjuvants useful herein are by definition chemical compounds added to vaccines to enhance the production of an immune response by the animal receiving the vaccine. Most adjuvants function by: (1)producing an irritation at the site of injection causing leukocytes (immune cells) to infiltrate the area, and/or (2) by producing a depot effect -- holding the antigen(s) at the injection site for as long as possible. If infiltration of leukocytes to the injection site is extensive, swelling and injection-site lesions will occur. Such leukocytes carry the antigens from the vaccine to cells within the immune system (of the vaccinated animal) which can produce a protective response. Some newer polymer adjuvants function by encapsulating antigens and releasing them slowly over a period of weeks or months.
  • the adjuvants can be selected from the group consisting of: oil-in-water, water-in-oil, Al(OH) 3 , Al 2 (SO 4 ) 3 , AlPO 4 , extracts of bacterial cell walls (Mycobacterium, Propionibacterium, etc.), extracts of plants (acemannan, saponin or Quil A), polymers, including block copolymers, liposomes and combinations thereof.
  • the adjuvants are polymers, including block copolymers (alternately referred to herein as polymer adjuvants.
  • a specific example of the preferred adjuvant is carbopol.
  • effective adjuvants can be formulated with the protective antigens to produce vaccines that are safe and effective.
  • a multicomponent vaccine for ruminants would include all the required protective antigen components and adjuvant, in a low dose. In essence, fewer than five protective antigens from each organism would be required to make a vaccine immunogenically effective. However, a vaccine containing only the protective antigens would be essentially a very pure vaccine. Because of the high purity of the antigens, it would be difficult adjuvant them with commonly used adjuvants. The pure antigen would require adjuvants that are different from the typical adjuvants. Therefore, a commercial scale production of clostridial vaccines containing very pure protective antigen components would be technically difficult. At any rate, the preparation of a very pure animal vaccine on a commercial scale is prohibitive because of the cost of purification.
  • individual components of the multicomponent vaccines described herein can be formulated with protective antigens derived from: whole culture bacteria, whole culture viruses, cell-free toxoids, purified toxoids and/or subunits.
  • Whole cultures contain numerous antigens. Some of the antigens impart protection (protective antigens), some produce negative response (detrimental antigens) and some are essentially neutral (neutral antigens).
  • Subunits can be obtained from the organisms themselves by conventional methods such as: centrifugation, ultrafiltration, and extraction with detergents or organic solvents. Alternately, the subunits can be produced by recombinant technology and expressed in live vectors or other organisms and isolated and purified. It would be understood that protective antigen components may contain few to many antigens at least one of which is protective or immunogenically effective.
  • the interference may result from: (1) physical masking or hiding of an active site of one protective antigen component by another, (2) aggregation or agglomeration of one or more protective antigen components so that one or more active sites are hidden from the immune system, (3) chemical interaction wherein there is a change in the active site of one protective antigen component by another. The latter change can result from a toxic effect, chemical binding or a conformational change in a critical portion of an active site.
  • the process comprises: using specialized procedures for identifying the protective antigen components; quantitating the protective antigen components; identifying those protective antigen components that contain detrimental antigens; purifying those protective antigen components that contain detrimental antigens to remove such detrimental antigens; selecting adjuvants that produce the necessary enhancement of the immune response without causing unacceptable reactivity and protect against interference ;individually adjuvanting the protective antigen components that are sensitive to the effects of detrimental antigens; pooling the various protective antigen components into a low dose volume vaccine.
  • the inventors employ adjuvant that protect the active sites of the various protective antigen components.
  • the adjuvants interact with targeted protective antigens, and not other antigens.
  • the selection of an adjuvant is critical.
  • the adjuvant must be one that is potent enough to produce significant enhancement of the immune response without producing unacceptable local or systemic reactions.
  • the term "produce significant enhancement of the immune response” refers to stimulation of the immune system such that protection of the host animal results from vaccination. Additionally, the adjuvant must reduce or prevent the interference with the protective antigens.
  • An adjuvant that encapsulates antigens is preferred. This characteristic is usually associated with polymer- or block copolymer-type adjuvants.
  • the preferred adjuvant for this invention is one containing "carbopol" or the equivalent thereof.
  • An integral part of the invention is the use of a specified test method for antigen quantitation of the protective antigen components.
  • the test method for quantitation of a clostridial protective antigen component involves injection of mice with combinations of antigen and specific antisera.
  • the test method is referred to herein as "a combining power test”.
  • the resultant measurement of antigen is designated as "combining power unit” (CPU).
  • the CPU test developed in accordance with the invention, is an integral part of the formulation of combination clostridial products.
  • the test comprises adding varying volumes of test material to a series of tubes.
  • test material in each tube is brought to 1.0 mL using Peptone Sodium Chloride Diluent [8.5 gm Sodium Chloride and 10 gm Bactone Peptone/liter (PND)].
  • PND Bactone Peptone/liter
  • the tubes are mixed and 18 to 20 gm mice are inoculated intravenously with 0.5 mL from each tube. The mice are observed for 48 hours and death is recorded.
  • Other test methods that produce substantially the same results as described herein are encompassed by the claimed invention.
  • Non-limiting examples of other test methods can be ELISA assays and liquid chromatography, which quantitate antigens directly in vaccines.
  • the skilled artisan can employ the required CPU/mL or the equivalent Elisa antigen quantitation unit to ascertain the value of the amounts of the antigenic components that are useful in making and using the vaccines of the invention.
  • multicomponent vaccines containing a plurality of clostridial protective antigen components plus at least one non-clostridial protective antigen component and an adjuvant in a low dose volume can be produced by: identifying the protective antigen component of each organism by in vivo or in vitro methods; quantifying the protective antigen components during formulation and manufacture of the vaccine, using antigen quantitation assays described above to provide the protective antigen component in an amount sufficient to produce a protective vaccine with the least antigenic mass; identifying the antigenic components of the organisms which contain detrimental antigens by using the antigen quantitation assays and animal reactivity testing; purifying the protective antigen components which contain detrimental antigens to remove such antigens; selecting the inactivating agent for each organism requiring inactivation such that the organism is killed without denaturing the protective antigen component; selecting an adjuvant for each protective antigen component that requires an adjuvant by evaluating the adjuvant's ability to enhance the immune response to the specific protective antigen component without
  • the multicomponent vaccine contains a combination of: one or more clostridial protective antigen components with one or more non-clostridial protective antigen components and an adjuvant within a low dose volume of 3.0 mL or less.
  • the use of multicomponent vaccines, i.e., commercial scale vaccines of this infection, do not produce significant injection-site lesions upon subcutaneous or intramuscular administration.
  • Cl. chauvoei protective antigens have been found by the inventors to be associated with cells These protective antigens are not found in proteinaceous material excreted into the culture supernatant while the organism is being grown in fermenters. It has also been found that the Cl. chauvoei protective antigen component does not interfere with other protective antigen components in the multicomponent clostridial vaccine. Therefore, a whole cell bacterin or a cell extract can be used. The whole cell bacterin or cell extract may be inactivated with formaldehyde (0.05 - 1.5%), Betapropriolactone (BPL) at 0.05 to 0.3% or Binary ethyleneimine (BEI) at 0.05 to 0.3%. After inactivation, this component must be adjuvanted separately.
  • formaldehyde 0.05 - 1.5%
  • Betapropriolactone BPL
  • BEI Binary ethyleneimine
  • Adjuvants which enhance this protective antigen component are Al(OH) 3 , oils, saponin, Quil A, block co-polymers and polymers such as "carbopol". Oil adjuvants can be used as co-adjuvants with polymers. Carbopol is more preferred and is added to the inactivated whole culture at a low pH. The pH is then adjusted upward to approximately 7.0 with, say, sodium hydroxide (NaOH). This pH adjustment step allows for the protective antigen components of the Cl. chauvoei to become encapsulated in the polymer adjuvant. Without being bound to any particular theory of the invention, it is believed the Cl. chauvoei antigens are released over a period of several weeks. Because of the slow release, these antigens do not cause the typical animal reaction. The long-term release causes an enhanced immune response by the vaccinated animal.
  • the protective antigen component of Cl. septicum is associated both with the cell and with a toxin.
  • the toxin is secreted into a supernatant while the organism is growing. Therefore, this protective antigen component is derived from the cell and supernatant.
  • Cl. septicum does not interfere with other protective antigen components in multicomponent clostridial vaccines containing non-clostridial protective antigen components.
  • the whole cell bacterin or cell extract can be inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%). After inactivation, this protective antigen component must be adjuvanted separately. When BPL or BEI are used for inactivation, they must be neutralized before adjuvanting.
  • Adjuvants that enhance this protective antigen component can be: Al(OH) 3 , oils, saponin, Quil A, block co-polymers and polymers such as carbopol. Oil adjuvants can be used if combined as co-adjuvants with polymers.
  • the preferred adjuvant are the polymer adjuvant.
  • the adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0 during which the antigens of the Cl. septicum become encapsulated in the carbopol. The resulting vaccine does not cause the typical animal reactivity but releases the Cl. septicum antigens over a period of several weeks. This mode of release causes an enhanced immune response by the vaccinated animal.
  • the protective antigen component of Cl. novyi is believed by the inventors to be associated with a cell protein, and a toxin that is excreted into a supernatant. Therefore, this protective antigen component is derived from both the cell and supernatant, in either concentrated or non-concentrated form. Apparently, the protective antigen of the Cl. novyi does not interfere with other protective antigen components in multicomponent clostridial vaccines when combined with non-clostridial protective antigen components.
  • the whole cell bacterin or cell extract may be inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%) and must be adjuvanted separately.
  • Adjuvants that enhance this protective antigen component are Al(OH) 3 , oils, saponin, Quil A, block co-polymers and polymers such as carbopol. Oil adjuvants can be used if combined as co-adjuvants with polymers.
  • the carbopol polymer adjuvants are preferred.
  • the polymer adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0 during which the antigens of the Cl. novyi become encapsulated in polymer.
  • the resulting vaccine does not cause the typical animal reactivity but releases the Cl. novyi antigens over a period of several weeks. The long-term release causes an enhanced immune response by the vaccinated animal.
  • the protective antigen component of Cl. sordellii is believed to be associated with a toxin that is secreted into the supernatant as the culture is growing. Therefore, this protective antigen component is derived from the supernatant. This protective antigen component is typically concentrated via ultrafiltration through a 10,000 dalton molecular weight (MW) cartridge before adjuvanting.
  • the Cl. sordellii toxin may be inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%) prior to adjuvanting, and must be adjuvanted separately. If BPL or BEI is used for inactivation, it must be neutralized before adjuvanting.
  • Adjuvants that enhance this protective antigen component are Al(OH) 3 , oils, saponin, Quil A, block co-polymers and polymers such as carbopol. Oil adjuvants can be used if combined as co-adjuvants with polymers.
  • the polymer adjuvant is are preferred.
  • the carbopol polymer adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0 during which the antigens encapsulated in polymer adjuvant.
  • the resulting vaccine does not cause the typical animal reactivity but releases the Cl. sordellii antigens over a period of several weeks. The long-term release causes an enhanced immune response by the vaccinated animal.
  • the protective antigen components of Cl. perfringens types C and D are known to be toxoids that are excreted by the cells. Because they cross-protect against Cl perfringens type B, these protective antigen components only need to contain cell-free supernatant containing inactivated toxin (toxoid). These two components are considered to represent 3 components (B,C, and D). In formulations of a multicomponent clostridial vaccine, one may use Cl. perfringens types C and D protective antigen components that contain cells or have the cells removed therefrom (cell free toxoid).
  • the whole culture is harvested from the fermenter and inactivated with formaldehyde (0.5-1.5%), BPL (0.05-0.5%) or BEI (0.05-0.5%) and before adjuvanting.
  • the cells can be removed by, say, filtration or centrifugation, In either case, the respective antigens must be adjuvanted separately. If BPL or BEI is used for inactivation, it must be neutralized before cell removal.
  • Adjuvants which enhance this protective antigen component are Al(OH) 3 , oils, saponin, Quil A, block co-polymers and polymers such as carbopol. Oil adjuvants can be used if combined as co-adjuvants with polymers. Preferred here is the polymer adjuvant.
  • the carbopol adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0. During this increase the protective antigen components of the Cl. perfringens become encapsulated in the polymer adjuvant.
  • the protective antigen component of Cl. haemolyticum is believed to be both cell-associated and excreted as a toxin into the supernatant. Therefore, this protective antigen component contains antigens from the cells and supernatant. Because of its high cell mass, this protective antigen component can cause interference with other protective antigen components of a multicomponent clostridial vaccine.
  • this protective antigen is concentrated by, say, ultrafiltration with a 10,000 molecular weight cartridge before adjuvanting.
  • the Cl. haemolyticum whole culture can be inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%) before concentration. The inactivated, concentrated material must be adjuvanted separately.
  • Adjuvants which enhance this protective antigen component are Al(OH) 3 , oils, saponin, Quil A, block co-polymers and polymers such as carbopol. Oil adjuvants can be used if combined as co-adjuvants with polymers. Preferred herein is the polymer adjuvant.
  • the carbopol adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0.
  • the protective antigen components of the Cl. haemolyticum become encapsulated in polymer adjuvant. The resulting vaccine does not cause the typical animal reactivity but releases the Cl. haemolyticum antigens over a period of several weeks. The long-term release causes an enhanced immune response by the vaccinated animal.
  • the multicomponent, low-dose vaccines can be administered subcutaneously or intramuscularly to protect animals without causing significant injection-site lesions.
  • This example illustrates the embodiment of this invention comprising a combination of protective antigen components from at least 6 clostridial organisms with protective antigen components from at least 1 non-clostridial component such as a Gram-negative organism.
  • a multi-component bacterin was formulated with a combination of protective antigen components derived from: Cl. chauvoei , Cl. septicum , Cl. novyi , Cl. sordellii , Cl. perfringens types C and D; a protective antigen component from H. somnus and a carbopol adjuvant.
  • the H. somnus protective antigen component was purified enough to prevent animal reactivity but not so much as to make it non-cost effective.
  • Each isolate was grown separately in 160 L of media containing the following components: Pancreatic Digest of Casein, Yeast Extract, Proteose Peptone, NaCl, and Na 2 HPO 4 .
  • the growth medium was supplemented with 0.5% dextrose and 10% horse serum. Dissolved oxygen was controlled during the fermentation cycle at approximately 10% (between 5% and 20%). Fermenters were inoculated with either 3.5% seed (isolate 14767) or 5% seed (isolate 8025T).
  • the whole bacterial cultures were concentrated 10X using a 0.1 micron ultrafiltration cartridge, followed by diafiltration with 11 volumes of Phosphate Buffered Saline (PBS).
  • PBS Phosphate Buffered Saline
  • the washed concentrates were then centrifuged at 7000 RPM using a Sorvall RC5B refrigerated centrifuge and the pellets were resuspended in 100 mL of chilled PBS. Centrifuged concentrates were adjusted to either 10X or 20X concentration (based on initial whole culture volume) and adjuvanted with 10% v/v 10X modified carbopol adjuvant.
  • This adjuvant was comprised of: up to 0.25% Carbopol 934P, Tween 80, Span 20 and Cotton Seed Oil.
  • H. somnus 8025T consisted of either 0.061 mL of adjuvanted H. somnus 8025T 20X concentrate or 0.122 mL of adjuvanted 10X concentrate.
  • a dose of H. somnus 14767 consisted of either 0.061 mL of adjuvanted 20X concentrate or 0.122 mL of adjuvanted 10X concentrate.
  • H. somnus preparations Relative purity of the above-described H. somnus preparations was demonstrated by comparing their endotoxin levels after the various purification steps. The preparations were compared to whole culture H. somnus . Samples of H. somnus 8025T and 14767 10X concentrates were removed at various stages in the purification process and diluted to 1X with PBS.
  • Endotoxin assays were run on the samples using an automated BioWhitaker apparatus and results were normalized against an E. coli LPS standard prepared to contain one million endotoxin units per mL. Results are shown in TABLE 1. Results show that the H. somnus cultures can be purified using centrifugation or a combination of ultrafiltration and diafiltration. The resultant cultures had endotoxin levels that were less than 10% of those seen in original inactivated whole cultures. This level of endotoxin reduction is adequate to eliminate significant animal reactivity and is cost effective. TABLE 1 ENDOTOXIN LEVELS OF PURIFIED H.
  • Example 1A illustrates that immunogenicity is maintained when only the cells were used to produce the protective antigen components.
  • the washed-cell preparations thereof were formulated at various antigen concentrations with a plurality of clostridial protective antigen components and tested as either a 2.0 mL dose or a 5.0 mL dose (positive control) in a mouse vaccination/challenge test [approved by the U.S. Animal Plant Health Inspection Service (APHIS)].
  • the test was conducted by vaccinating mice with a fractional dose of the test product, boostering such mice with the same dose at 14 days post vaccination and challenging such mice with a virulent H. somnus culture at 10-14 days post booster.
  • the challenge culture was mixed with an equal volume of 7% gastric mucin prior to injection.
  • the resulting mixture was strong enough to kill 80% of the control mice (16 of 20). For a satisfactory test, at least 14 of 20 vaccinated mice must survive.
  • the clostridial fractions were produced as follows:
  • any commercial Cl. chauvoei whole bacterial culture could be used as the protective antigen component, for purposes of this experiment the Cl. chauvoei was grown under strict anaerobic conditions in large-scale fermenters under pH control conditions between 6.5 and 7.6; inactivated with 0.5% formaldehyde and adjuvanted with the modified carbopol adjuvant as a separate non-concentrated whole bacterial culture.
  • the modified carbopol adjuvant was the same as that described in Example 1A.
  • the adjuvant was added in a 10% v/v ratio to the Cl. chauvoei whole bacterial culture, mixed to allow complete contact with adjuvant while at a low pH, and then pH adjusted to approximately 7.0 with 5 or 10N NaOH.
  • the Cl. septicum was grown under strict anaerobic conditions in large-scale fermenters with pH control between 6.5 and 7.6; inactivated with 0.5% formaldehyde, concentrated minimally using a 10,000 dalton MW ultrafiltration system and adjuvanted with the modified carbopol adjuvant by adding the adjuvant directly to the concentrated whole bacterial culture Cl. septicum .
  • the modified carbopol adjuvant is the same as that described previously.
  • the adjuvant was added in a 10% v/v ratio to the Cl. septicum concentrate, mixed to allow complete contact with adjuvant at the low pH, and then pH adjusted to approximately 7.0 with 5 or 10N NaOH.
  • Cl. novyi was grown under strict anaerobic conditions in large-scale fermenters with pH control between 6.5 and 7.6, inactivated with 0.5% formaldehyde and adjuvanted as a non-concentrated whole bacterial culture with the modified carbopol adjuvant as described previously.
  • the adjuvant was added in a 10% v/v ratio to the Cl. novyi whole bacterial culture, mixed to allow complete contact with adjuvant at low pH, and then pH adjusted to approximately 7.0 with 5 or 10N NaOH.
  • Combining Power Unit (CPU) was measured, as described above, in the culture post inactivation and post adjuvanting.
  • the CPU of the final protective antigen component was adjusted to 10 CPU/mL with adjuvanted PBS.
  • Cl. sordellii was grown under strict anaerobic conditions in large-scale fermenters with pH control between 6.5 and 7.6. At the end of the growth phase, the culture was maintained at a pH of approximately 8.0 for 8-10 hours to facilitate cell lysis. The lysed culture was then inactivated with 0.5% formaldehyde (lysed toxoid), concentrated using a 10,000 dalton MW ultrafiltration cartridge and adjuvanted with the modified carbopol adjuvant described previously. The adjuvant was added in a 10% v/v ratio to the Cl. sordellii lysed toxoid, mixed to allow complete contact with adjuvant at the low pH, and then pH adjusted to approximately 7.0 with 5 or 10N NaOH. After adjuvanting, the combining power was measured and the protective antigen component was adjusted to 100 CPU/mL by dilution with adjuvanted PBS.
  • Clostridium perfringens types C and D were grown under strict anaerobic conditions in large-scale fermenters with pH control between 7.3 and 7.5 for 4-8 hours. The whole bacterial cultures were inactivated with 0.5% formaldehyde. For purposes of this experiment, cells were removed by centrifugation in a Sorvall centrifuge at 7000 RPM. The remaining supernatants contained Cl. perfringens C or D toxoids. The toxoids were individually concentrated by ultrafiltration through a 10,000 dalton MW cartridge and the concentrates were assayed for their quantity of protective antigen component by the previously-described combining power test.
  • each protective antigen component was individually adjuvanted using the modified carbopol adjuvant described previously.
  • the adjuvant was added in a 10% v/v ratio to the individual Cl. perfringens toxoids (C or D), mixed to allow complete contact with adjuvant at the low pH, and then pH adjusted to approximately 7.0 with 5 or 10N NaOH.
  • Cl. haemolyticum was grown under strict anaerobic conditions in large-scale fermenters with pH control between 6.8 and 7.3. The culture was harvested and inactivated with 0.5% formaldehyde prior to concentration. A 10,000 dalton MW ultrafiltration cartridge was used to concentrate the whole culture which was then adjuvanted with the modified carbopol adjuvant described in Example 1A. The adjuvant was added in a 10% v/v ratio to the Cl. haemolyticum culture concentrate, mixed to allow complete contact with adjuvant at low pH, and then pH adjusted to approximately 7.0 with 5 or 10N NaOH.
  • H. somnus was prepared according to the description in Example 1A.
  • H. somnus protective antigen component was still potent when the washed cells were resuspended to a concentration equal to one-half the concentration of the original whole culture and mixed with 6 clostridial protective antigen components.
  • Cl. haemolyticum was added to the 6 original clostridial protective antigen components it appeared to adversely affect the H. somnus protective antigen component only slightly - not enough to require a dose size greater than 2.0 mL.
  • This example shows the effect of detrimental antigens on relatively weak protective antigen components such as C. perfringens types C and D.
  • the effect of the detrimental antigens were evaluated in a multicomponent vaccine containing protective antigen components from 6 clostridial organisms and one protective antigen component from one non-clostridial .
  • Clostridial protective antigen components were produced as described in Example 1B.
  • Serials were formulated with varying levels of Cl. perfringens type C and D toxoids.
  • CPU levels for type C were adjusted to 600, 900, 1200 or 1800 per dose whereas CPU levels of type D toxoid were adjusted to 350, 500, 700 or 1000 per dose.
  • the five multicomponent clostridial vaccines and one vaccine containing a plurality of clostridial protective antigen components combined with H. somnus were tested according to procedures required by the U.S. government Animal Plant Health Inspection Service (APHIS). Guinea pigs, rabbits or mice were used for the testing. For the clostridial components, guinea pigs or rabbits were vaccinated respectively with a dose equivalent to 1/5 or 1/2 the field dose. These animals were boostered 10 to 14 days later with the same dose of vaccine. Guinea pigs were challenged with live organisms of either Cl. chauvoei or Cl. haemolyticum . To correlate with protection in cattle, at least 80% of the guinea pigs must survive these challenges.
  • mice were vaccinated, boostered and challenged to demonstrate that a vaccine was protective against H. somnus .
  • the challenge was a live culture of H. somnus which must kill at least 80% of the non-vaccinated control mice. An acceptable vaccine must protect 14 of 20 vaccinated mice.
  • Rabbits were vaccinated, boostered and bled to test for antibody titers against Cl. septicum , Cl. sordellii , Cl. novyi , and Cl. perfringens types C and D.
  • Antibody quantitation was conducted according to prescribed APHIS testing against known standard toxins and antitoxins.
  • This example shows the incorporation of the protective antigen components from the clostridial organisms and H. somnus in a commercial size serial of a vaccine, and the test for potency of the components.
  • a 160 L batch of 6-way clostridial product containing Cl. chauvoei , Cl. septicum , Cl. novyi , Cl. sordellii , Cl. perfringens types C and D was prepared in the proportions as listed in TABLE 4 and formulated as in Example 2 with H. somnus isolates 8025T and 14767 at a 1X concentration as described in Example 1A.
  • This serial was tested for potency according to the previously-described APHIS requirements. The results of the tests are shown in TABLE 6.
  • perfringens type C/B 3602 540 NONE 0.400 mL Cl. perfringens type D/B 455E 155 NONE 0.364 mL H. somnus 8025T N/A 20X 0.061 mL H. somnus 14767 N/A 20X 0.061 mL
  • Adjuvanted PBS N/A N/A N/A 0.056 mL TABLE 8 ANIMAL TEST RESULTS PRODUCED BY 7-WAY + H. somnus ORGANISM TEST ANIMAL TYPE OF TEST REQUIREMENT FOR SATISFACTORY POTENCY POTENCY RESULT 7-WAY+ H. somnus Cl.
  • This example illustrates vaccines wherein viruses are combined with clostridial components.
  • Modified live infectious bovine rhinotracheitis virus IBRV
  • a plurality of clostridial protective antigen components Cl. perfringens types C and D.
  • the clostridial protective antigen components were prepared and formulated according to methods discussed in Example 1B.
  • the IBRV utilized for this experiment was one which had been modified such that it would a disease if the live virus is injected into animals. Vaccines prepared from such viruses are called modified live vaccines. Since modified live vaccines contain live viruses as their protective antigen component, the efficacy of such vaccines depends on the amount of live virus contained within them. It has been determined by cattle vaccination/challenge studies that infectious bovine rhinotracheitis virus when prepared in a lyophilized vaccine protects cattle if the titer is at least 10 4.2 TCID 50 /mL.
  • the reference IBRV used for this experiment was grown in roller bottle culture on bovine kidney cells after which the IBRV harvest fluids were lyophilized such that the titer post lyophilization was 10 7.0 /mL.
  • the multicomponent vaccine containing protective antigen components from Cl. perfringens types C and D and from IBRV is formulated as a two-container vaccine.
  • One container will contain the lyophilized modified live IBRV protective antigen component and the second container will contain the inactivated, adjuvanted liquid Cl. perfringens types C and D protective antigen components.
  • the liquid Cl. perfringens types C and D protective antigen component is removed from its container with a syringe and injected into the lyophilized modified live IBRV container causing rehydration of the lyophilized IBRV.
  • APHIS defines viricidal activity as the loss of more than 0.7 logs of virus titer within 2 hours after rehydrating the virus component. Any multicomponent vaccine in which the virus protective antigen component loses more than 0.7 logs of virus titer within 2 hours post rehydration by the diluent therefore would be considered to have failed the viricidal activity test.
  • CELL-FREE TOXOID 400 10 7.5 +0.5 12x894-B NONPURIF.
  • CELL-FREE TOXOID 700 10 7.0 0.0 12X894-C PURIF.
  • CELL-FREE TOXOID 600 PURIF.
  • CELL-FREE TOXOID 400 10 7.0 0.0 12X894-D PURIF.
  • CELL-FREE TOXOID 550 10 6.9 -0.1 12X894-E PURIF.
  • CELL-FREE TOXOID 700 10 6.7 -0.3
  • the reference titer for the IBRV rehydrated with sterile diluent was 10 7.0 .
  • This example shows that a larger combination of virus protective antigen components and clostridial protective antigen components could be successfully prepared in a low dose formulation.
  • Several preparations of Cl. perfringens types C and D protective antigen components were prepared as described in Example 1B and combined with modified live IBRV, modified live bovine virus diarrhea virus (BVDV), modified live parainfluenza type 3 virus (PI 3 ) and modified live bovine respiratory syncytial virus (BRSV).
  • the four modified live virus protective antigen components were prepared by art-known techniques. As part of the preparation, the detrimental effect of the clostridial protective antigen components on any of the modified live virus protective antigen components was determined.
  • the APHIS-required viricidal activity test was conducted on the various multicomponent vaccines. Since clostridial vaccines historically contain residual formaldehyde as a preservative and since it is known that formaldehyde can have a detrimental effect on modified live viruses, part of this experiment involved adding known amounts of formaldehyde to the formulations to determine maximum allowable amounts of this preservative. TABLE 11 lists the formulation differences and the results of the viricidal activity testing for the four virus protective antigen components. The results indicate that the clostridial protective antigen components are somewhat viricidal especially to IBRV and BVDV.
  • Two sources of yearling cattle were randomly allocated to treatment groups of 54 head each.
  • Two-milliliter dose 6-way clostridial vaccine (formulated as in Example 2) was given subcutaneously to one group and 5.0 mL dose, 6-way vaccines formulated via conventional methods but containing the modified carbopol adjuvant was administered subcutaneously to the other group.
  • the cattle were commingled throughout the trial. Evaluations of the injection sites were made on days 7, 21, 49 and 95 days post injection. Results are shown in Figures 1 and 2. On day 7, all animals had a palpable injection site response in both groups.
  • calves with a known injection history were used to evaluate the incidence and duration of injection site lesions in carcasses from animals injected intramuscularly.
  • the calves were at branding and weaning age.
  • a 5.0 mL dose conventional 6-way clostridial product or a 2.0 mL dose 6-way clostridial multicomponent vaccine prepared by the methods of this invention were administered in the semimembranosus muscle (inside round steak location) at branding using an 18 gauge, 1-inch needle.
  • a 2.0 mL dose vaccine containing 6 clostridial protective antigen components combined with protective antigen components from H. somnus was prepared according to the methods described in Example 2 and administered to 1,528 calves by six veterinarians in five states.
  • the field trial was conducted from November 1994 through January 1995.
  • Vaccine was administered by the normal routes of administration for the herd and included both intramuscular and subcutaneous routes. Veterinarians were requested to observe the calves for injection site reactions and/or lesions. At the end of the trial, no significant unfavorable local or systemic reactions were noted by any of the participating veterinarians.
  • a multicomponent vaccine containing protective antigen components from at least 6 clostridial organisms, protective antigen components from at least one non-clostridial organism such as a Gram-negative bacteria like H. somnus and an adjuvant such as carbopol can be produced commercially in a dose volume less than 3.0 mL and safely injected to protect animal under field conditions.

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EP0841392A3 (fr) * 1996-11-12 1999-03-24 Pfizer Inc. Neospora vaccin vivant atténué
EP0924295A3 (fr) * 1997-12-04 2001-05-16 Pfizer Products Inc. Gène de dihydrofolate reductase-thymidilate synthase de neospora
US6787146B2 (en) 1997-08-26 2004-09-07 Pfizer Inc. Neospora vaccine

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US6743430B1 (en) * 1995-03-29 2004-06-01 Richard E. Parizek Multicomponent vaccine containing clostridial and non-clostridial organisms in a low dose
FR2768747B1 (fr) * 1997-09-19 2000-12-01 Pasteur Institut Acides nucleiques, cellules recombinantes, et procede de preparation de compositions immunogenes
AU2007245198B2 (en) * 2006-03-30 2010-08-12 Zoetis Services Llc Methods and compositions for vaccination of poultry
CN102648003B (zh) * 2009-10-09 2016-01-13 儿童医疗中心有限公司 选择性裂解的全细胞疫苗
BR112012010223A2 (pt) 2009-10-30 2016-12-06 Novartis Ag purificação de staphylococcus aureus tipo 5 e tipo 8 sacarídeos capsulares
AR086199A1 (es) 2011-04-22 2013-11-27 Wyeth Llc Composiciones relacionadas con una toxina de clostridium difficile mutante y sus metodos
BR122016023101B1 (pt) 2012-10-21 2022-03-22 Pfizer Inc Polipeptídeo, composição imunogênica que o compreende, bem como célula recombinante derivada de clostridium difficile
EP4168032A2 (fr) 2020-06-19 2023-04-26 Pfizer Inc. Compositions immunogènes contre clostridioides (clostridium) difficile et méthodes associées

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US3790665A (en) * 1968-02-23 1974-02-05 Haver Lockhart Labor Inc Injectable adjuvant,method of preparing same and compositions including such adjuvant
US3925544A (en) 1971-07-12 1975-12-09 Univ Southern Illinois Bovine vaccines and methods of making and using same
US3920811A (en) * 1972-12-22 1975-11-18 Cutter Lab Adjuvant compositions
SU627610A1 (ru) 1977-09-30 1980-02-05 Zemlyakova V P Препарат землаково в.п. против клюстридиозов животных и птиц
NZ193807A (en) 1979-05-31 1983-03-15 Ici Tasman Ltd Improving the response of ruminants to clostridial vaccine by administering tetramisole or levamisole
WO1994022476A1 (fr) * 1993-03-29 1994-10-13 Pfizer Inc. Vaccins composes de constituants multiples derives de clostridium et comportant des adjuvants a base de saponine
US6743430B1 (en) * 1995-03-29 2004-06-01 Richard E. Parizek Multicomponent vaccine containing clostridial and non-clostridial organisms in a low dose

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0841392A3 (fr) * 1996-11-12 1999-03-24 Pfizer Inc. Neospora vaccin vivant atténué
US6787146B2 (en) 1997-08-26 2004-09-07 Pfizer Inc. Neospora vaccine
EP0924295A3 (fr) * 1997-12-04 2001-05-16 Pfizer Products Inc. Gène de dihydrofolate reductase-thymidilate synthase de neospora

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